WO2020076136A9 - Akkermansia muciniphila strain and use thereof - Google Patents

Abstract

The present invention relates to a Akkermansia muciniphila SNUG-61027 strain (accession number KCTC 13530BP) and a use thereof. Specifically provided are: a composition for appetite control or prevention, improvement, alleviation or treatment of metabolic diseases, containing, as an active ingredient, the strain, a culture liquid thereof and the like, or a B2UM07 protein isolated therefrom; a use of the composition for appetite control or prevention, improvement, alleviation and treatment of metabolic diseases; and a method for appetite control or prevention, improvement, alleviation and treatment of metabolic diseases by using the composition. Therefore, the present invention exhibits effects of weight loss and glucose homeostasis control, from among anti-obesity effects, and influence on brown fat and secretion of an appetite control hormone secretion.

Description

Akermancia muciniphila strain and use thereof

Cross-reference with related application(s)

This application claims the interests of priority based on Korean Patent Application No. 10-2018-0121137 filed October 11, 2018 and Korean Patent Application No. 10-2019-0125670 filed October 10, 2019. All contents disclosed in the documents are included as part of this specification.

The present invention relates to the Akermansia muciniphila SNUG-61027 strain (accession number KCTC 13530BP) having an appetite suppression and metabolic disease prevention, amelioration, amelioration and treatment efficacy, and a use thereof.

Obesity is a state in which excessive body fat is accumulated in the human body due to changes in dietary habits such as high-calorie diets, lack of exercise, etc., and is associated with the onset of type 2 diabetes, cardiovascular disease, liver disease, and various cancers, and is of great clinical importance. On the other hand, intestinal microbes are known to have a profound correlation with metabolic diseases such as obesity and diabetes. In particular, Akermansia muciniphila strain increases in the intestine of mice treated with meformin, a diabetes treatment, and this strain is used as a high-fat diet mouse. As it was found that glucose homeostasis improved when administered to, it is attracting attention as a treatment for obesity, suggesting a new paradigm for anti-obesity drug research.

Various studies have been conducted to understand the mechanism of the anti-obesity effect of the Akermansia muciniphila strain, whose anti-obesity efficacy has been verified, among intestinal microbes close to 10 times the total number of human cells. The focus is on weight loss, improving chronic metabolic inflammation, restoring damaged barriers, or improving blood lipid levels.

However, it has been recently reported that the anti-obesity effect has various mechanisms in addition to the above-mentioned indicators, and in particular, the induction of brown fat interacts with intestinal microbes in connection with the body temperature maintenance homeostasis mechanism. Adipose tissue is divided into white fat, which stores energy in the form of triglycerides, and brown fat, which releases energy as heat. Brown fat regulates glucose homeostasis and insulin sensitivity by inducing energy consumption through tissue-specific UCP-1 factor. It has a function that enhances.

On the other hand, glucagon-like peptide (GLP-1), an appetite regulating hormone, is a hormone secreted from the small intestine by food intake, increasing satiety, controlling appetite, and inducing insulin secretion from the pancreas to regulate blood sugar. do.

GLP-1 is secreted from L-cells, a type of intestinal endocrine cells present in the ileum and colon.

The GLP-1 is a diabetes treatment effect, obesity treatment effect, heart disease treatment effect, cerebrovascular disease and nerve cell inflammation treatment effect (Salcedo I et al., Neuroprotective and neurotrophic actions of glucagon-like peptide-1 (GLP-1): an emerging opportunity to treat neurodegenerative and cerebrovascular disorders.British Journal of Pharmacology (2012) 166, 1586-1599), atherosclerosis treatment effect (Burgmaier M et al., Glucagon-like peptide-1 (GLP-1) and its split products) GLP-1(9-37) and GLP-1(28-37) stabilize atherosclerotic lesions in apoe-/- mice.Atherosclerosis (2013) 231, 427-435).

In addition, GLP-1 stimulates glucose-dependent insulin secretion in the pancreas, enhances insulin gene expression, increases pancreatic beta cell proliferation, enhances pancreatic beta cell survival, inhibits glucagon secretion, lowers blood sugar, etc. It is involved in slowing the rate of emptying of the stomach, suppressing appetite, promoting satiety, and inhibiting food intake, thereby exhibiting the effect of treating obesity. In addition, it has the effect of protecting cardiomyocytes from ischemia and treating heart disease through the effect of improving cardiac function in patients with a risk of heart attack (Sokos, GG etal., Glucagon-like peptide-1 infusion improves left ventricular). ejection fraction and functional status in patients with chronic heart failure.J. Card. Fail. (2006) 12: 694-699., Ban, K., etal., Cardioprotective and vasodilatory actions of glucagon-like peptide-1 receptor are mediated through both glucagon-like peptide-1 receptor-dependent and -independent pathways.Circulation (2008) 117: 2340-2350.).

GLP-1 activates TGR5 and GPR119, a kind of G protein-coupled receptors (GPCRs) (Reimann, F., etal., Glucose sensing in L cells: a primary cell study. Cell Metab. (2008) 8:532) -539; Lauffer, LM, et al., GPR119 is essential for oleoylethanolamide-induced glucagon-like peptide-1 secretion from the intestinal enteroendocrine L-cell. Diabetes (2009) 58:1058-1066) or activation of α-gustducin (Jang , HJ, etal., 2007. Gut expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1.Proceeding of the National Academy of Science 104, 1506915074.). In particular, activation of G protein-coupled receptor (GPCR) TGR5 (GPR131) expressed in brown adipose tissue and muscle increases energy expenditure, thereby showing an effect of treating obesity, and is related to improvement of liver disease. (Lieu T et al., GPBA: AG protein-coupled receptor for bile acids and an emerging therapeutic target for disorders of digestion and sensation. British Journal of Pharmacology (2013) in press), and has been shown to inhibit arteriosclerosis. It has been reported (Pols TWH et al., TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation.Cell Metabolism (2011) 14, 747).

In addition, triglycerides accumulated excessively in obese patients are stored not only in adipose tissues, but also in the liver or muscles to induce insulin resistance. Therefore, the consumption of excessively stored triglycerides can be the prevention and treatment of underlying obesity and metabolic diseases. Adipocytes are largely classified into white fat, brown fat cells and beige fat cells. White adipose cells are stored in large fat cells of triglycerides and are mainly found in the abdomen and are known to play a negative role in health. Brown fat contains more mitochondria and small-sized fat cells than white fat cells, and it has been reported that it can be induced by maintaining body temperature through heat generation and proper exercise. Mice induced to contain large amounts of brown fat cells were effective against obesity and metabolic diseases by inducing weight loss and increased caloric consumption relative to obesity caused by a high fat diet. In addition, a large amount of UCP-1 (uncoupling protein-1) protein is expressed in brown fat, which is known to play a crucial role in the generation of heat by consuming calories rather than storing calories in adipocytes. In addition to brown fat, beige fat cells are also recognized as important fat cells. Beige adipocytes are induced by stimulation such as exercise or cold in white adipocytes, which are harmful to health, and the traits of white adipocytes decrease, but have the characteristic of brown adipocytes, thereby increasing the expression of UCP-1. Their beige adipocytes are also known to be beneficial for obesity and metabolic diseases similar to the brown adipocytes found in mice.

[Prior technical literature]

1. Korean Patent Registration No. 10-1809172

2. Korean Patent Publication No. 10-2015-0133646

Under this background, the present inventors are the standard strain currently used in anti-obesity studies for the purpose of effectively preventing and treating metabolic diseases, Akkermansia muciniphila , Akk ; American strain bank, accession number: ATCC BAA-835) and Akermancia muciniphila SNUG-61027 strain isolated from healthy Korean feces (accession number: KCTC13530). It was confirmed to increase the expression of the appetite control hormone GLP-1 in the small intestine.

In addition, the present inventors have shown that this process is induced dependently on the IL-6 cytokine of the host, and finally, Akermansia muciniphila strain culture solution, cells, supernatant, which promote the secretion of GLP-1 in the anti-obesity mechanism, The present invention was completed by identifying the extract or fraction thereof, or the target protein derived from the strain.

As an aspect for achieving the above object, the present invention provides an accession number KCTC 13530BP of Akkermansia muciniphila ( Akkermansia muciniphila , Akk ) SNUG-61027 strain. Specific information on the strain is as follows.

Name of donated institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC 13530BP

Consignment Date: 20180525

The strain of the present invention includes 16S rDNA consisting of the nucleotide sequence of SEQ ID NO: 1.

In addition, the present invention provides a pharmaceutical composition for preventing, improving or treating appetite suppression or metabolic diseases using the Akermansia muciniphila SNUG-61027 (accession number KCTC 13530BP) strain or a culture solution thereof as an active ingredient.

The term "culture solution" of the present invention is obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that Akermansia muciniphila SNUG-61027 (accession number KCTC 13530BP) strain can grow and survive in vitro. It refers to the whole medium including the strain, its metabolites, and extra nutrients, but it is a concept including all the supernatant from which the strain is removed after culturing the strain, extracts and fractions thereof. The liquid from which the cells have been removed from the culture medium is also referred to as "supernatant," and the culture medium is left for a certain period of time to take only the liquid of the upper layer excluding the submerged part, or remove the cells through filtration, or the culture medium is centrifuged to precipitate the lower part. It can be obtained by removing and taking only the upper liquid.

The "bacterial body" refers to the strain of the present invention itself, and includes the strain itself selected by separating from fermented food or the strain isolated from the culture medium by culturing the strain. The cells can be obtained by centrifuging the culture medium to take a portion that has settled in the lower layer, or because it sinks to the lower layer of the culture medium by gravity, it can be obtained by leaving it for a certain time and then removing the upper liquid.

In addition, the extract of the Akermansia muciniphila SNUG-61027 (accession number KCTC 13530BP) strain culture, cells or supernatant of the present invention is an extract extracted with ethyl acetate (EtOAc) or ethanol (ethanol, ethyl alcohol; EtOH) It may be, but is not limited thereto. Further, the fraction of the Akermancia muciniphila SNUG-61027 strain culture solution, cells, or supernatant of the present invention may be a fraction obtained by fractionating the ethyl acetate extract with methanol, but is not limited thereto. Akermancia muciniphila (accession number KCTC 13530BP) strain culture medium, supernatant or fraction of the extract of the present invention can be obtained according to a conventional fractionation method well known in the art, for example, anion exchange column or size column. It can be obtained by a chromatography method using

The term “metabolic disease” of the present invention means that one or two or more diseases, such as impaired glucose tolerance, diabetes, fatty liver, hypertension, dyslipidemia, obesity, and cardiovascular atherosclerosis, appear in one individual due to chronic metabolic disorders. And, for example, it may be any one selected from impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease, and obesity.

By the present invention, an increase in IL-6, an increase in GLP-1 expression, and an increase in the activity of brown fat may be induced to have a beneficial effect on the metabolic disease, and further, the metabolic disease may be prevented, improved or treated.

The present invention provides a pharmaceutical composition for preventing, improving or treating appetite suppressing or metabolic diseases comprising the B2UM07 protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

The B2UM07 protein was identified through NCBI Database matching of the existing strain when performing the protein identification in the efficacy fraction of the present invention through LC/MS-MS, and the information is as follows.

Gene: Amuc_1631

UniProtKB-B2UM07

Protein name-Carboxyl-terminal protease

Organism: Akkermansia muciniphila

The B2UM07 protein may be derived from an Akermancia muciniphila strain, and specifically, the Akermancia muciniphila strain may be SNUG-61027 strain (accession number KCTC 13530BP).

In addition to the protein consisting of the amino acid sequence of SEQ ID NO: 2, the present invention is considered to be included within the scope of the present invention. A variant is a protein consisting of an amino acid sequence consisting of a base sequence having functional properties similar to that of SEQ ID NO: 2, although the base sequence or amino acid sequence is changed. Specifically, the protein according to the present invention is an amino acid having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the amino acid sequence of SEQ ID NO: 2 Sequence.

In addition, the present invention provides a gene encoding the B2UM07 protein. The gene of the present invention includes both genomic DNA and cDNA encoding the B2UM07 protein, respectively. Preferably, the gene may include a nucleotide sequence encoding the protein of SEQ ID NO: 2.

In addition, variants of the base sequence are included within the scope of the present invention. Specifically, the gene has a sequence homology of 60% or more, more preferably 70% or more, even more preferably 80% or more, most preferably 90% or more with the base sequence encoding the protein of SEQ ID NO: 2 It may include a base sequence.

Another aspect of the present invention provides a recombinant vector comprising the gene encoding the B2UM07 protein according to the present invention. The term "recombinant" of the present invention refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells may express genes or gene segments that are not found in the natural form of the cell in either a sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in a natural state, but the gene is modified and reintroduced into the cell by artificial means.

The "vector" is used when referring to a DNA fragment(s) or nucleic acid molecule to be delivered into a cell. Vectors replicate DNA and can be reproduced independently in host cells. In addition, the present invention provides a transformant transformed with the recombinant vector. As a method of transforming the vector into E. coli, a method commonly known in the art such as the use of a competent cell using a CaCl ₂ buffer, electroporation, and heat shock can be used. have. The method of culturing the transformed E. coli may use a culture method of E. coli commonly used in the art.

The pharmaceutical composition according to the present invention can be administered to mammals including humans by various routes. The term "administration" of the present invention means introducing a predetermined substance to an individual by an appropriate method, and the mode of administration may be any method commonly used, for example, oral, skin, intravenous, intramuscular, subcutaneous routes, etc. It can be administered as, preferably orally. The pharmaceutical compositions of the present invention may be used in oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., or parenteral formulations such as ointments, aerosols, transdermals, suppositories and sterile injectable solutions according to a conventional method. It can be formulated and used in a formulation or the like. The pharmaceutical composition of the present invention may further contain adjuvants such as a pharmaceutically suitable and physiologically acceptable carrier, excipient, and diluent.

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium Silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used.

In an embodiment in which the pharmaceutical composition of the present invention is applied to humans, the pharmaceutical composition of the present invention may be administered alone, but generally, a pharmaceutical composition selected in consideration of the mode of administration and standard pharmaceutical practice. It can be administered in admixture with a carrier. For example, the composition containing the strain of Akermancia muciniphila of the present invention is in the form of a tablet containing starch or lactose, alone or in the form of a capsule containing an excipient, or a chemical agent that gives taste or color. It can be administered orally, orally or under the tongue in the form of containing elixirs or suspensions.

The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease degree, and according to the judgment of a doctor or pharmacist, once a day or several times a day It can also be administered in divided doses. For example, the daily dosage may be 0.1 to 500 mg/kg, preferably 0.5 to 300 mg/kg, based on the content of the active ingredient. The above dosage is an example of an average case, and the dosage may be high or low depending on individual differences.

Another example of the present invention is Akermancia muciniphila strain SNUG-61027 (accession number KCTC 13530BP) or a culture medium, supernatant, extract or fraction thereof as an active ingredient for suppressing appetite or improving or alleviating metabolic disease health Provide functional food.

The metabolic disease may be impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease, or obesity.

According to the present invention, an increase in IL-6, an increase in GLP-1 expression, and an increase in the activity of brown fat may be induced to exhibit a beneficial effect on the metabolic disease, and further, the metabolic disease may be alleviated or treated.

In addition, the present invention provides a health functional food for suppressing appetite or improving or alleviating metabolic diseases, including the B2UM07 protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

The metabolic disease may be impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease, or obesity.

The B2UM07 protein may be derived from an Akermancia muciniphila strain, and specifically, the Akermancia muciniphila strain may be SNUG-61027 strain (accession number KCTC 13530BP), and specific details are as described above. same.

The health functional food may be various beverages, fermented milk, food additives, and the like.

The content of the Akermansia muciniphila strain as an active ingredient contained in the health functional food is not particularly limited appropriately depending on the form of the food and the desired use, and may be added, for example, in 0.01 to 15% by weight of the total food weight. In addition, the health beverage composition may be added in a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

Among the health functional foods of the present invention, there is no particular limitation on liquid components other than those containing the Akermancia muciniphila strain as an essential component in the indicated ratio as an essential component, and various flavoring agents or natural carbohydrates, etc. May contain as an additional component.

Examples of the above-described natural carbohydrates are monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose, and the like, and polysaccharides such as dextrin, cyclodextrin, and the like. These are sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid, and It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.

In addition, the health functional food of the present invention may contain flesh for the manufacture of natural fruit juice and fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not so important, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the health functional food of the present invention.

The present invention is Akermansia muciniphila ( Akkermansia muciniphila ) SNUG-61027 strain (accession number KCTC 13530BP), or a culture medium, supernatant, extract or fraction thereof to suppress appetite or use for prevention, treatment, improvement or alleviation of metabolic diseases Provides.

The Akkermansia muciniphila SNUG-61027 strain (accession number KCTC 13530BP) used in the use of the present invention may contain 16S rDNA consisting of the nucleotide sequence of SEQ ID NO: 1.

The metabolic disease to which the use of the present invention is applied may be impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease or obesity.

In addition, the present invention provides a use of the B2UM07 protein consisting of the amino acid sequence of SEQ ID NO: 2 for preventing, treating, improving or alleviating appetite or metabolic diseases.

The B2UM07 protein used in the use of the present invention may be derived from Akermancia muciniphila strain.

The Akermancia muciniphila strain used in the use of the present invention may be SNUG-61027 strain (accession number KCTC 13530BP).

The present invention is Akermansia muciniphila ( Akkermansia muciniphila ) SNUG-61027 strain (accession number KCTC 13530BP), or a culture medium, supernatant, extract or fraction thereof, including the step of treating appetite suppression or metabolic disease prevention, treatment, Provides a way to improve or mitigate.

The Akkermansia muciniphila SNUG-61027 strain (accession number KCTC 13530BP) used in the method of preventing, treating, ameliorating or alleviating appetite suppression or metabolic disease of the present invention consists of the nucleotide sequence of SEQ ID NO: 1. It may include 16S rDNA.

The metabolic disease to which the method of the present invention is applied may be impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease, or obesity.

The present invention provides a method for preventing, treating, ameliorating or alleviating appetite suppressing or metabolic diseases comprising the step of treating the B2UM07 protein consisting of the amino acid sequence of SEQ ID NO: 2.

The B2UM07 protein used in the method of the present invention may be derived from Akermancia muciniphila strain.

The Akermancia muciniphila strain used in the method of the present invention may be SNUG-61027 strain (accession number KCTC 13530BP).

The present invention confirms the activating effect of brown fat and the ability to secrete the appetite-regulating hormone GLP-1 in addition to weight loss and glucose homeostasis among the anti-obesity effects of Akermansia muciniphila, and these effects are found in the host's specific cytokine, IL-6. Confirmed that it is dependent. In addition, in the present invention, a novel Akermansia muciniphila SNUG-61027 strain (accession number KCTC 13530BP) having significantly increased GLP-1 induction ability was discovered, and B2UM07 (P9) isolated from the Akermansia muciniphila strain culture solution It was confirmed that the protein exhibited remarkably excellent GLP-1 induction ability, ability to maintain glucose homeostasis in the body, and weight loss effect. Therefore, the novel Akermancia muciniphila strain and B2UM07 protein may be usefully used for suppressing appetite or treating or preventing metabolic diseases.

1 is a result of an experiment on the effect of improving liver and brown fat weight after administration of Akkermansia muciniphila ( Akk ) strain to a high fat diet mouse model.

2 is an experimental result confirming the increase of UCP-1 expression and brown fat-related markers by the Akermansia muciniphila strain using qPCR.

3 is an experimental result confirming an increase in IL-6 cytokine and an increase in GLP-1 in the small intestine by Akermansia muciniphila strain using qPCR.

4 is an experimental result confirming that brown fat expression and exothermic reaction by Akermansia muciniphila strain is dependent on IL-6 cytokine.

Figure 5 is in vitro This is an experimental result confirming that the expression of GLP-1 by Akermancia muciniphila is caused by a bacterial secretion substance (ELISA).

6 is an experimental result confirming that the expression of GLP-1 by Akermancia muciniphila in vitro is caused by factors other than short-chain fatty acid (SCFA) (GC-MS).

Figure 7A is an experimental result confirming the GLP-1 expression ability by the size fraction of Akermansia muciniphila in vitro, Figure 7B is a proteolytic enzyme (proteinase K; PK) in the GLP-1 derived fraction (100K, 300K) ) After treatment, the results of the experiment confirming the expression of GLP-1.

8 shows the results of fractionation and GLP-1 derived fractionation experiments of the GLP-1 derived fraction (100K) using an anion exchange column and a size column.

9 is a result of qualitative analysis of the protein derived from Akermancia muciniphila GLP-1 fraction (100K, m2-m4, G17-G20) using LC/MS-MS.

10 is an experimental result confirming the ability to induce GLP-1 by a purely purified candidate protein (SDS-PAGE gel).

11 is an experimental result confirming the ability of glucose homeostasis in the body when the target protein is administered intraperitoneally.

12 is an experimental result confirming the ability of glucose homeostasis in the body when the target protein is administered orally.

Hereinafter, the present invention will be described in more detail by the following examples or experimental examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1.Akermancia muciniphila ( Akkermansia muciniphila , Akk) analysis of liver and brown fat weight reduction effect after administration

Akermancia muciniphila (ATCC BAA-835, Akk) strain was anaerobicly cultured in a brain heart infusion (BHI) solid medium supplemented with 0.5% mucin for 72 hours, and then stock was obtained. Six-week-old male C57BL/6 mice were orally administered a high-fat diet (60% fat) feed at a concentration of 4 x 10 ⁸ CFU/200 µl/mouse daily (HF+Akk, n=8/group). A group that consumed only a low-fat diet (10% fat) diet (LF) and a high-fat diet (HF) was used as a control group. After 14 weeks, the control group and the strain administration group were compared. After fasting for 16 hours, adipose tissue and liver tissue were collected and the tissue weight was measured (Fig. 1A).

As a result, there was no significant change in the weight of inguinal white adipose tissue (igWAT) and epididymal white adipose tissue (EpiWAT) in the HF+Akk group compared to the HF group, but interscapular brown adipose tissue. tissue, iBAT) weight significantly decreased. In addition, when the correlation analysis of brown fat (iBAT) and liver tissue weight with body weight was performed (Fig. 1B), it was confirmed that the brown fat and liver tissue weight was significantly proportional to the body weight, thereby reducing brown fat and liver weight. Suggested the possibility of being a target tissue of Akermancia muciniphila.

In addition, as a result of comparing the fat size of brown adipose tissue and liver tissue by group through hematoxylin eosin staining (H&E staining), the fat size of brown adipose tissue and liver tissue was remarkably in the Akermansia muciniphila group compared to the control group. It was observed to decrease (Figs. 1C and D).

Therefore, as a result of the experiment, it was confirmed that Akermansia muciniphila contributes to a reduction in the weight and fat size of brown adipose tissue and a reduction in the weight of liver tissue (FIGS.

Example 2. Increased UCP-1 expression and brown fat-related markers by Akermansia muciniphila strain

In brown adipose tissue (iBAT), uncoupling protein (UCP-1), which is a brown fat activation marker, was subjected to immuno-histochemistry (IHC) staining and compared for each group (FIG. 2A). After tissue RNA was extracted and cDNA synthesized, the gene expression of UCP-1 was confirmed by qPCR, and markers related to brown adipose differentiation (CIDEA, PRDM16, PPARGC1α, Apelin) were also confirmed (FIG. 2B).

As a result of the experiment, it was confirmed that the brown adipose-related markers of the brown adipose tissue were significantly increased in the high-fat-inducing mice administered with Akermansia muciniphila compared to the non-administered group, and the UCP-1 factor involved in the brown fat activity was stained with tissue. In one result, an increase was confirmed, confirming the mechanism of brown fat induction of Akermancia muciniphila.

Example 3. Increased IL-6 cytokine and GLP-1 in the small and large intestine by Akermansia muciniphila strain

After extracting RNA from small intestine and colon tissue, synthesizing cDNA, the level of expression of immune cytokine markers (TNF-α, IL-1β, IL-18, IL-6, IL-10) It was compared by group (Fig. 3A,B).

When treating mouse intestinal cell line (CT26 cell) with three Lactobacillus (KCTC2180, KCTC3112, KCTC1048) and Bifidobacterium three (KCTC3127, KCTC3128, KCTC3352) or Akermansia muciniphila (Akk), IL-6 cytotoxicity The expression ability of kine was compared. Lipopolysaccharide (LPS) from E. coli was used as a positive control (Fig. 3C).

Related genes (gcg, pcsk1, pcsk2) inducing the expression of intestinal secreted appetite regulating hormone, glucagon-like peptide-1 (GLP-1) in the small intestine tissue were confirmed by qPCR (Fig. 3D).

As a result of the experiment, IL-6 cytokine was significantly increased in the small intestine and colon cells of the mouse by administration of Akermansia muciniphila, and the expression of the appetite regulating hormone, glucagon-like peptide-1 (GLP-1) in the serum was significantly increased. It was confirmed that it increased significantly (Figs. 3A to 3D). In particular, Akermancia muciniphila in mouse intestinal cell lines showed significantly increased IL-6 levels compared to other Lactobacillus and Bifidobacterium strains.

Example 4. Whether brown fat expression and exothermic reaction by Akermansia muciniphila strain is dependent on IL-6 cytokine

It was confirmed whether Akermansia muciniphila's brown fat activation effect was dependent on IL-6 cytokine.

To this end, 6-week-old male C57BL/6 Wild type (WT) mice and IL-6 gene deficient mice (IL-6KO) mice were fed with a high-fat diet (60% high fat; HF) and 4 x 10 strains at the same time. ^It was orally administered daily at a concentration of ⁸ CFU/200 μl/mouse (n=6 in the IL-6KO group, n=8/group in the other groups). A group that consumed only a low-fat diet (10% low fat; LF) and a high-fat diet was used as a control group. After 14 weeks, WT mice and IL-6KO mice were compared with the high fat diet group and the strain administration group.

After fasting for 16 hours, the brown adipose tissue was isolated, RNA was extracted, cDNA was synthesized, and UCP-1 expression was confirmed by qPCR (Fig. 4A). The rectal temperature was measured using a digital thermometer (TESTO925) (FIG. 4B). The skin temperature of the brown fat was measured using a thermal imaging camera (FLIR) (FIGS. 4C and D).

To measure the concentration of GLP-1 in the serum, after inducing fasting in the morning for 5 hours, glucose was administered orally at a concentration of 2g/kg. After 10 minutes, plasma was collected through orbital blood collection and placed in a cold-maintained tube containing 1 µg/ml diprotin A (6019; Tocris), which suppresses the half-life of GLP-1. After centrifugation (4,000 X g , 10 min), the supernatant was frozen at -80°C. Thereafter, GLP-1 expression was measured through the mouse GLP-1 ELISA kit (Fig. 4E).

Related genes (gcg, pcsk1, pcsk2) that induce GLP-1 expression in small intestine (ileum) tissues and colon tissues were confirmed by qPCR in WT mice and IL-6KO mice (FIGS. 4F-H).

As a result of the experiment, the expression of the brown fat-related gene UCP-1, whose expression was increased by administration of Akermancia muciniphila, was not increased in IL-6 gene deficient mice (Fig. 4A). In addition, even when the skin surface temperature of the brown fat area was measured with an infrared camera or an anal thermometer, it was confirmed that heat generation due to brown fat activation did not appear in IL-6KO mice (FIGS. 4B to 4D). In addition, unlike WT mice, the concentration of GLP-1 in serum was rather reduced in IL-6KO mice, and there was no change in the level of genes (gcg, pcsk1, pcsk2) that induces the expression of GLP-1, so the appetite regulating hormone GLP in the small intestine It was confirmed that the increase of -1 was also dependent on IL-6 (FIGS. 4E-H).

Example 5. It was confirmed that the expression of GLP-1 by Akermancia muciniphila was caused by a bacterial secretion substance ( in vitro )

After culturing the Akk strain (Akermancia muciniphila ATCC BAA-835) or Akermancia muciniphila SNUG-61027 strain in 0.5% mucin medium, 0.1% or 5% Fetal bovine serum ( FBS) was incubated in BHI medium added for 36 hours.

After seeding the NCI-H716 (ATCC CCL-251) cell line secreting GLP-1 in a collagen-coated 96-well plate at a concentration of 2 x 10 ⁵ cells/ml, synchronization of cellular metabolism to glucose between cells was performed. For this, 0.2% bovine serum albumin (BSA) was incubated with HBSS (Hanks Buffered Saline Solution) for 2 hours. Thereafter, Akk strain (ATCC BAA-835) or Akermansia muciniphila SNUG-61027 bacterial pellet (strain to cell ratio: 1:20) or culture supernatant (cell free supernatant; CFS) 10% v/ Treated with v concentration. After 2 hours, the supernatant was obtained, and the level of GLP-1 expression in the supernatant was measured using an ELISA kit (FIG. 5A). In order to confirm the GLP-1 expression efficacy by concentration, the culture supernatant of the SNUG-61027 strain was added at a concentration of 10 to 100% v/v or as a control (con) in the same manner as above. The derived strain) was treated at a concentration of 10-100% v/v, and then 2 hours later, a supernatant was obtained to confirm the expression of GLP-1 in the supernatant (FIG.

As a result of the experiment, when live cells and supernatant of Akermansia muciniphila were treated with GLP-1 induced cell line (L cells), GLP-1 was not measured during treatment with live cells, whereas GLP- It was confirmed that 1 was highly expressed, and the expression level was significantly increased in SNUG-61027 strain than ATCC BAA-835 (FIG. 5A). In addition, when the culture supernatant of the SNUG-61027 strain was treated, the expression level of GLP-1 was increased in a concentration-dependent manner (Fig. 5B).

Example 6. It was confirmed that the expression of GLP-1 by Akermancia muciniphila was caused by factors other than short-chain fatty acids ( in vitro )

Expression of representative short chain fatty acids, acetate, propionate, and butyrate was confirmed using GC-MS for the analysis of Akermansia muciniphila secreted short chain fatty acid (SCFA). (Fig. 6A). Two hours after treatment with acetate and propionate (1 mM, 10 mM) and strain culture supernatant (100% v/v), the level of GLP-1 expression was confirmed (FIG. 6B).

As a result of the experiment, it was confirmed that Akermancia muciniphila secretes acetate and propionate (FIG. 6A). However, GLP-1 induced by acetate and propionate was significantly less than GLP-1 expressed by the culture supernatant of Akermancia muciniphila (Fig. 6B). Therefore, it was found that GLP-1 induced by Akermancia muciniphila is involved in elements other than acetate and propionate.

Example 7. Fractionation of GLP-1 derived fraction (100K) using size filter, anion exchange column and size column and identification of GLP-1 derived fraction

In order to separate the active material in the culture medium, each fraction was obtained using a filter according to size. Thereafter, after concentrating it, it was confirmed that a high level of GLP-1 was expressed in a fraction between 100kDa and 300kDa when the ability to induce GLP-1 was confirmed. In addition, in order to remove the protein in the effective fraction (100K~300K, 30K~100K), proteinase K (PK) was treated at a concentration of 100 ㎍/㎖ for 1 hour at 55℃, and then inactivated at 90℃ for 10 minutes. Afterwards, GLP-1 expression was measured. As a result, it was confirmed that GLP-1 expression was not induced in the fraction from which the protein was removed, and through this, it was confirmed that GLP-1 was induced by the protein in the fraction. Fast protein liquid chromatography (FPLC) using a MonoQ anion exchange column (MomoQ 5/50, GE Healthcare) and AKTAexplorer system (GE Healthcare) to re-fraction 100kDa~300kDa (100K) of the Akermancia muciniphila supernatant. ) Proceeded. A 100K fraction of 80 μg/ml was injected, and the samples were fractionated at a rate of 1 ml/min. Thereafter, each fraction was treated with L cells, and the level of GLP-1 expression was measured. As a result of the experiment, it was confirmed that a high content of GLP-1 was expressed in the m2-m4 fraction (FIG. 8A).

Thereafter, the m2-m4 fraction was concentrated with a 30K filter, and the concentrated sample was again subjected to FPLC using a GPC size column (GPC/SEC). For fractionation, the sample was fractionated at a rate of 3 ml/min using a hiload 16/600 Superdex pg (GE Healthcare) AKTAexplorer system. In the same way, each fraction was treated on L cells, and the ability of GLP-1 expression was confirmed. As a result of the experiment, it was confirmed that GLP-1 was expressed at a high level in the G17-G20 fraction (FIG. 8B).

Example 8. Qualitative analysis of Akermansia muciniphila GLP-1 derived fraction (100K, m2-m4, G17-G20) protein using LC/MS-MS

Sample 1) 100K concentrate, Sample 2) MonoQ concentrate, Sample 3) GPC concentrate obtained from the Akermansia muciniphila supernatant were analyzed qualitatively through LC/MS-MS. Bovine-related proteins that can be found in the basal medium of the supernatant were excluded, and the number of proteins identified in each fraction was confirmed.

To this end, a qualitative analysis was performed on each sample obtained through a size filter, and finally, 10 kinds of proteins or peptides expressed in the GPC concentrate that were supposed to have been concentrated were listed by intensity, and compared with the level of expression in other fractions. LC-MS/MS (Nanoflow Easy-nLC 100/Q Exactive mass spectrometer) analysis instrument was used. The processing was performed using Maxquant software 1.5, and the protein was qualitatively analyzed by annotation using the Universal Protein Resource (Uniprot) protein database. For total proteins and peptides, only those with a false discovery rate <1% were selected.

As a result of the experiment, 10 proteins were identified in Sample 3) G17-G20 fraction, which is considered to be the most concentrated candidate protein (Fig. 9A,B).

Example 9. Confirmation of GLP-1 induction ability by purely purified candidate protein

After cloning, 10 kinds of proteins derived from the concentrated fraction of Akermansia muciniphila were expressed in E.coli BL21 cells to purely isolate the protein. One of the 10 proteins (Beta-galactosidase) was excluded in the steps below because an effective expression vector could not be obtained by cloning. After that, it was confirmed that 9 kinds of proteins separated after expression were purely separated through SDS-PAGE. Amuc1100 is a protein with a known anti-obesity function derived from Akermansia muciniphila (Plovier H. et al ., A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice. Nat Med. (2017) 23 :107-113) Used as a positive control. Each isolated protein was treated on L cells to confirm GLP-1 expression.

To this end, the synthesized target protein was inserted into a pET-21b plasmid (Novagen) with an IPTG inducing promoter, and then purified purely through his-tag. This was confirmed through SDS-PAGE gel. The synthesized plasmid was transformed into BL21 Escherichia coli strain and cultured to mass-produce and isolate the protein, and after measuring the protein concentration, it was treated in the NCI-H716 cell line .

As a result of the experiment, interestingly, it was confirmed that the expression of GLP-1 was induced by the proteins B2UKW8(P1), B2URM2(P5), and B2UM07(P9). It was confirmed that GLP-1 was induced at a significantly higher level (FIG. 10C).

Example 10. Confirmation of glucose homeostasis ability of target protein in the body (normal diet, intraperitoneal administration)

In order to confirm whether the in vivo glucose homeostasis ability is improved by the discovered target protein, P1 (B2UKW8), P5 (B2UKW8), P9 (B2UM07) proteins were administered intraperitoneally to a normal diet mouse at a concentration of 100 μg/mouse for a week, The sugar resistance test was conducted.

To this end, 3 kinds of efficacy proteins (P1, P5, P9) were administered abdominal to normal diet mice at a concentration of 100 µg/200 µl daily, and 7 days later, the body weight was compared with the non-administered group (n=8/group , FIG. 11C) , 14 days after administration, after oral administration of glucose at a concentration of 2g/kg, blood glucose was measured for 15 minutes to 120 minutes to conduct a glucose tolerance test by time (FIGS. 11A and 11B ).

As a result of the experiment, it was confirmed that the P9 (B2UM07) administration group maintained significantly lower blood sugar than the other groups. This was shown to be more effective than Amuc1100 protein derived from Akermancia muciniphila, which is known to have glucose tolerance. P1 (B2UKW8) and P5 (B2UKW8) only showed a tendency toward glucose tolerance, but in the case of the P9 group, weight loss was also significantly confirmed (FIG. 11C).

Example 11. Confirmation of the ability of target protein to homeostasis in the body (high fat diet, oral administration)

In order to confirm whether the in vivo glucose homeostasis ability was improved by the discovered target protein, the P9(B2UM07) protein was administered orally to high-fat diet mice at a concentration of 100 µg/mouse for 8 weeks, and then a glucose tolerance test was performed. Blood glucose was measured for 15 to 120 minutes after oral administration of glucose (2 g/kg).

As a result of the experiment, the P9 (B2UM07) administration group showed a significant weight gain inhibitory effect compared to the high fat diet mouse group, and the effect was greater than that of the Amuc1100 administration group (FIG. 12A). In addition, 30 minutes after glucose administration, it was confirmed that the glucose homeostasis ability was significantly regulated compared to the high fat diet mouse group (FIGS. 12B and 12C).

Claims (28)

1. Akkermansia muciniphila SNUG-61027 strain (accession number KCTC 13530BP).
2. According to claim 1, wherein the strain is Akermansia muciniphila SNUG-61027 strain (accession number KCTC 13530BP), characterized in that it comprises a 16S rDNA consisting of the nucleotide sequence of SEQ ID NO: 1.
3. A pharmaceutical composition for preventing, improving or treating an appetite suppressing or metabolic disease comprising the Akermansia muciniphila SNUG-61027 strain of claim 1, or a culture medium, supernatant, extract or fraction thereof as an active ingredient.
4. The pharmaceutical composition for preventing, improving or treating appetite suppression or metabolic disease according to claim 3, wherein the metabolic disease is impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease or obesity.
5. The method of claim 3, wherein the Akermancia muciniphila SNUG-61027 strain, or a culture solution, supernatant, extract or fraction thereof, increases IL-6, increases GLP-1 expression, and increases the activity of brown fat. A pharmaceutical composition for preventing, improving or treating appetite suppression or metabolic disease, characterized in that to induce.
6. A pharmaceutical composition for preventing, improving or treating appetite suppressing or metabolic diseases, comprising the B2UM07 protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.
7. The pharmaceutical composition of claim 6, wherein the B2UM07 protein is derived from Akermancia muciniphila strain.
8. The pharmaceutical composition for preventing, improving or treating appetite suppressing or metabolic diseases according to claim 7, wherein the Akermancia muciniphila strain is SNUG-61027 strain (accession number KCTC 13530BP).
9. The pharmaceutical composition of claim 6, wherein the metabolic disease is impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease, or obesity.
10. Akermancia muciniphila SNUG-61027 strain of claim 1, or a health functional food for suppressing appetite or improving or alleviating metabolic diseases, comprising the culture medium, supernatant, extract or fraction thereof as an active ingredient.
11. The health functional food according to claim 10, wherein the metabolic disease is impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease, or obesity.
12. The method of claim 10, wherein the Akermancia muciniphila SNUG-61027 strain, or a culture solution, supernatant, extract or fraction thereof, increases IL-6, increases GLP-1 expression, and increases the activity of brown fat. Health functional food for suppressing appetite or improving or alleviating metabolic diseases, characterized in that to induce.
13. Health functional food for suppressing appetite or improving or alleviating metabolic diseases, comprising the B2UM07 protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.
14. The health functional food for suppressing appetite or improving or alleviating metabolic diseases according to claim 13, wherein the B2UM07 protein is derived from Akermancia muciniphila strain.
15. The health functional food according to claim 14, wherein the Akermancia muciniphila strain is SNUG-61027 strain (accession number KCTC 13530BP).
16. [14] The dietary functional food of claim 13, wherein the metabolic disease is impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease, or obesity.
17. Akkermansia muciniphila ( Akkermansia muciniphila ) SNUG-61027 strain (accession number KCTC 13530BP), or a culture medium, supernatant, extract or fraction thereof to suppress appetite or prevent, treat, ameliorate or alleviate metabolic diseases.
18. The appetite suppression or metabolic disease according to claim 17, wherein the Akkermansia muciniphila SNUG-61027 strain (accession number KCTC 13530BP) comprises 16S rDNA consisting of the nucleotide sequence of SEQ ID NO: 1 For the prevention, treatment, improvement or alleviation of
19. The use of claim 17, wherein the metabolic disease is impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease, or obesity. .
20. Use of B2UM07 protein consisting of the amino acid sequence of SEQ ID NO: 2 for suppressing appetite or preventing, treating, improving or alleviating metabolic diseases.
21. [21] The use of claim 20, wherein the B2UM07 protein is derived from Akermancia muciniphila strain.
22. The use of claim 21, wherein the Akermancia muciniphila strain is SNUG-61027 strain (accession number KCTC 13530BP), characterized in that it is an appetite suppression or metabolic disease prevention, treatment, improvement or alleviation.
23. Akermansia muciniphila ( Akkermansia muciniphila ) SNUG-61027 strain (accession number KCTC 13530BP), or a culture medium, supernatant, extract or fraction thereof, including the step of treating appetite suppression or metabolic disease prevention, treatment, improvement or alleviation Way.
24. The appetite suppression or metabolic disease of claim 23, wherein the Akkermansia muciniphila SNUG-61027 strain (accession number KCTC 13530BP) comprises 16S rDNA consisting of the nucleotide sequence of SEQ ID NO: 1 Prevention, treatment, improvement or alleviation of the method.
25. The method of claim 23, wherein the metabolic disease is impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolosis, fatty liver, cardiovascular disease, or obesity.
26. A method for preventing, treating, improving or alleviating appetite suppressing or metabolic diseases, comprising the step of treating the B2UM07 protein consisting of the amino acid sequence of SEQ ID NO: 2.
27. The method of claim 26, wherein the B2UM07 protein is derived from Akermansia muciniphila strain.
28. The method of claim 27, wherein the Akermansia muciniphila strain is SNUG-61027 strain (accession number KCTC 13530BP). The method of preventing, treating, improving or alleviating appetite suppressing or metabolic diseases.

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